1. Field of the Invention
The invention is directed to an optical arrangement for photomanipulation of a sample comprising a sample holder for receiving the sample, an illumination device comprising an illumination light source and an illumination beam path for illuminating the sample with a light sheet, a detection device for detecting light that is radiated from the sample, imaging optics which image the sample at least partially on the detection device by means of an imaging objective in an imaging beam path, wherein the light sheet is substantially planar in the focus of the imaging objective, and wherein the imaging objective has an optical axis which intersects the plane of the light sheet at an angle different from zero, preferably perpendicularly, a control unit, and means for photomanipulation of the sample.
The optical arrangement according to the invention can be applied for observation of the sample particularly in connection with single plane illumination microscopy (SPIM), also known as selective plane illumination microscopy. Whereas in confocal laser scanning microscopy the sample is scanned point by point in a plurality of planes at different depths and three-dimensional image information about the sample is obtained from this, the SPIM technique is based on widefield microscopy and makes it possible to generate three-dimensional images of samples based on optical sections through different planes of the sample.
The advantages of SPIM include faster acquisition of images, reduced bleaching out of biological samples, and an expanded depth of penetration of the focus in the sample.
2. Description of Related Art
Basically, in the SPIM technique fluorophores which are contained in the sample or introduced into the sample are excited by laser light which is shaped as a light sheet or which is guided over the sample in such a way that the shape of a light sheet results in effect, i.e., over the period of observation. Each light sheet illuminates a plane in the depth of the sample, and an image of the sample in this plane is obtained by means of this illumination. It is important that elements in the light sheet plane are projected on the detector plane or that the light sheet plane and detector plane are conjugate to one another. In conventional microscope constructions in which the detector plane extends perpendicular to the optical axis of the detection beam path, the direction in which light is detected is perpendicular, or at least virtually perpendicular, to the plane of illumination.
SPIM technology is described, for example, in Stelzer et al., Optics Letter 31, 1477 (2006), Stelzer et al., Science 305, 1007 (2004), DE 102 57 423 A1, and WO 2004/0530558 A1.
Aside from the observation of samples, the manipulation of biological, living or non-living matter and inorganic matter is also highly important in microscopy. For example, many samples can be photoactivated, photodeactivated, or heated. Manipulation also includes the processing of samples, i.e., for example, the polymerization of samples or, for example, the separation of areas of the sample from the rest of the sample by means of laser scalpels. Manipulation methods based on the interaction of light with the sample to be manipulated, e.g., laser ablation, bleaching, photoactivation, are particularly suitable when mechanical contact with the sample is to be avoided. Areas can be manipulated very precisely at microscopic resolution with appropriately designed optics.
For samples having a substantially flat shape such as, e.g., adherent cells or interfaces, manipulation can also be carried out very precisely with optics which do not offer high resolution axially.
However, for spatially extensive, three-dimensional samples, spatially precise manipulation is very difficult and can only be accomplished with complicated setups which are also often based on nonlinear interactions between the manipulation light beam and the sample.
Some of the standard techniques of photomanipulation-fluorescence loss in photobleaching (FLIP), photoactivation (PA GFP), reversible photoactivation (Dronpa), photoconversion (kaede), fluorescence localization after photobleaching (FLAP), microdissection, uncaging, to name only a few examples—have already been known and established in microscopy for decades. Usually, a laser beam of suitable power and wavelength is focused via an observation objective and directed to the sample. With corresponding power modulation, the laser beam can also be used for excitation for applications in fluorescence microscopy. Manipulation techniques, for example, the LASEC technique, are also used in ophthalmology.
However, there is a limited availability of devices which can carry out a specific manipulation in three-dimensional samples and which allow three-dimensional imaging of the sample at the same time. Confocal laser scanning microscopes are usually used for this purpose. However, the manipulation is not confocal with respect to excitation, even when this is true for the imaging. Therefore, the sample area that is illuminated is appreciably larger than is actually necessary. Only scanning microscopes using nonlinear interactions, e.g., two-photon excitation, offer a solution for this purpose. In microscopes of this kind, a confocal manipulation area which can be positioned in the sample with some accuracy is also formed on the excitation side. However, solutions of this type involve very complicated technology; for example, they require the use of short-pulse lasers and are limited with respect to choice of wavelength.
The manipulation of samples using microscopes is described, for example, in DE 102 33 549.